Understanding Retinogenesis and Fate Choice during Retinal Development

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In the study of this membrane, I for the first time felt my faith in Darwinism weakened, being amazed and confounded by the supreme constructive ingenuity revealed in the retina ... I felt more profoundly than in any other subject of study the shuddering sensation of the unfathomable mystery of life.

Santiago Ramón y Cajal 1898

The mouse retina offers an ideal system to study the molecular mechanisms of development since retinal cells are easily accessible, well characterized and develop in a defined sequence. The retina comprises six types of neurons and one type of glial cell, all of which can be identified by their position, morphology and by specific cell markers. All retinal cells are derived from a population of multipotent progenitors. Several necessary obstacles must be overcome during development for effective processing of visual information. The cell types must form in the proper ratios and in a specific order. They must migrate to a specific layer, differentiate and form synaptic connections. (Swaroop, Kim and Forrest, 2010) The fidelity of these processes can only be upheld if the mechanisms governing them are under tight regulation.

The cellular and molecular mechanisms in place for regulating the major aspects of retinogenesis have not yet been fully elucidated. It is clear however, that they involve both intrinsic and extrinsic factors which act upon the progenitor cells to control differentiation and direct changes in competence over developmental time. Quote The inductive interactions are slowly being worked out through analysis based upon gene expression patterns, immunohistochemistry and by loss of function mice. A complex interplay of transcription factors including basic helix-loop-helix and homeodomain proteins, have been identified that act as regulators of this developmental cascade. (Hatakeyama, 2004) These have been an important starting point for understanding the signaling systems and regulatory pathways responsible for differentiation.

The development of the photoreceptors has been a major focus in this line of research, as they are the most numerous in the retina. They are easy to access as they are in the outer nuclear layer (OLN), and are some of the last cells to develop in sequence. They also hold the greatest promise for clinical applications such as retinal replacement therapies for visual disorders. The study by Brzezinski, Lamba and Reh focuses on the differentiation of photoreceptors and bipolar cells. These cell types are similar in that they both express Otx2, a homeodomain transcription factor, which has been determined essential for the cell fate determination of photoreceptor cells and plays a functional role in the maturation of bipolar cells. (Rowan and Cepko, 2004) Bipolar cells reside in the inner nuclear layer (ILN) of the retina and are responsible for integration of signals. They became interested in the mechanism by which cells containing Otx2 chose between these two fates. It has already been shown that Chx10, a homeobox-containing transcription factor is critical for bipolar cell determination in the developing retina, and can repress photoreceptor development (Livne-bar, 2006) The interplay between these two transcription factors was unclear and could result various factors such as the molecular context altering target specificity, regulatory consequences, or competence of retinal progenitors. Blimp 1, a Prdm family transcriptional repressor was of interest as it has a fundamentally important role in the development of many cell types, and its expression has been reported in developing retinal precursors (Chang, Cattoretti and Calmane, 2002)

To investigate the role of Blimp1 in vivo, the researchers immunostained tissues at various times in the prenatal retina and used microscopy and PCR to determine the range of relative expression of Blimp1 and various other transcription factors during retinogenesis in wild type mice. They found that Blimp1 and Otx2 were always co-expressed in developing photoreceptors by embryonic day 12 and that Otx2 is expressed before Blimp1. The investigators produced conditional knockout (CKO) mice using Cre-recombinase to determine the role of Blimp1 in developing photoreceptors. Three and four week old CKO mice retinas showed fewer photoreceptors in the OLN and a thicker INL. Staining for markers revealed the thicker INL was due to an increase in bipolar cells. The bipolar cells were irregularly arranged. Because the loss of photoreceptors was four times greater than the gains in bipolar cells the researchers began to investigate whether this was due to photoreceptor apoptosis or increased bipolar genesis. It was clear at this point however, that Blimp1 somehow regulates cell fate choice between the two cell types. Brzezinski, Lamba and Reh began to investigate photoreceptor markers in early developmental stages and found unusually early cell markers for bipolar cells in the Blimp1 CKO retina, suggesting that the Otx2 cells were differentiating into bipolar cells. To investigate the time course of these differences, cell counts using staining in postnatal day seven mice were done. A decrease in Otx2 photoreceptors was seen to correspond exactly with the gain in bipolar with no difference in total Otx2 counts between the CKO and controls. This implied a one to one fate shift.

The accumulated data indicated that Blimp1 works to inhibit the development of bipolar cells in the mouse retina. To test if this is sufficient; a gain of function experiment was undertaken involving the construction of a Blimp1 green fluorescent protein (GFP) plasmid and in-vivo electroporation at a time where the fate choice in Otx2 cells occurs. The cells were given adequate time for the fate change to occur, and by labeling it was seen that Blimp1 GFP transfected cells took on a photoreceptor fate to a much greater degree than bipolar cells when compared to the GFP controls.

Loss of function data revealed a shift in fate choice in the opposite direction of the gain of function data. Combined, the experiments allowed Brzezinski, Lamba and Reh to reasonably conclude that the role of Blimp1 in regulating cell fate choice is by inhibiting the bipolar cell developmental pathway in Otx2 photoreceptor precursors. The data indicates that Blimp1 does not specify, but stabilizes photoreceptor cell fate during development. There was some data not consistent with this conclusion however. In some of the Blimp1 CKO mice photoreceptors still developed. This raises the possibility that another Prdm family member or unknown factor exists to compensate for the loss of Blimp1, or that Blimp1 is produced briefly in all Otx2 cells and not specifically in those resulting in photoreceptors. Further analysis of the regulatory pathways must be done, possibly by microarray analysis to detect upregulation of another such factor. Investigation into the latter possibility would require the establishment of cell fate maps via genetic lineage tracing. A third possibility is that there is some epigenetical regulation in this developmental cascade which requires studies involving chromatin immunoprecipitation, in situ hybridization or bioinformatics as examples.

There are some limitations to a study such as this one. For instance, while the manipulation of individual factors provides important and useful information, a more integrated description of the microenvironmental influences that regulate differentiation requires the development of new strategies to examine how networks of factors influence each other. Second, mouse studies have been necessary to examine retinogenesis, but caution should be exercised in using the results as the basic foundation for a general model of photoreceptor cell fate specification in vertebrates. The mouse retina Much research effort in the near future must be devoted to determining whether specific genetic mechanisms of photoreceptor determination and differentiation are conserved among species. (Adler, 2005)

Investigating the mechanisms of retinal development is fundamentally important to gaining a basic knowledge into how vision is established, and as degeneration of retinal neurons leads to irreversible vision loss. Future therapies may include retinal regeneration. Although the generation of stem cells is now routine, the development of reliable protocols for directed differentiation is imperative before the clinical regeneration of something as complex and patterned as the retina will be possible. (Poss, 2010) Transplantation of photoreceptors is feasible, provided donor cells are at an appropriate stage of development when transplanted. (McLaren et al, 2006)

Nevertheless, the proportion of cells that integrate into the recipient ONL is low. It could be that the mature retina is not able to promote photoreceptor differentiation. The study by Brzezinski, Lamba and Reh contributes a small piece of the puzzle, but an important one. Once the pathways that govern the differentiation of each cell type are made clear, this resource will revolutionize laboratory cell biology and will provide much improved cell culture models for the discovery and development of drugs and treatments for blindness, and fundamental studies of the genetic basis of disease.

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